Phosphorothioate triester oligonucleotides and method of preparation

ABSTRACT

PCT No. PCT/FR94/00563 Sec. 371 Date Jan. 17, 1996 Sec. 102(e) Date Jan. 17, 1996 PCT Filed May 11, 1994 PCT Pub. No. WO94/26764 PCT Pub. Date Nov. 24, 1994The trister phosphorothioate oligonucleotides disclosed comprise internucleotide concatenations having a P-S- bond protected by a bioreversible grouping (X) in intracellular media.

The present invention relates to phosphorothioate triesteroligonucleotide compounds and to a method of preparation.

In the present application, the term "oligonucleotides" generallyspeaking indicates a DNA or RNA polynucleotide, that is to say one inthe ribo- (RNA) or deoxyribo- (DNA), or even mixed ribo-deoxyribo,series. These oligonucleotides are in general formed by a linkage of 2to 100 nucleotides, and more generally, of 5 to 50 nucleotides.

These oligonucleotides are used for biological or therapeutic purposesaccording to different approaches: antisense (formation of duplex),anti-gene (formation of triple helixes), catalytic (RNA with ribozymeactivity) or sense (protein target).

The antisense oligonucleotides are short synthetic DNA or RNA or mixedmolecules of complementary sequences to a target sequence belonging to agene or to an RNA messenger whose expression it is specifically desiredto block. The antisense oligonucleotides are in fact directed against amessenger RNA sequence, or alternatively against a DNA sequence andhybridize to the sequence to which they are complementary, thus beingable to block genetic expression.

The antisense deoxyribonucleotides can also be directed against certainbicatenary DNA regions (homopurine/ homopyrimidine sequences orsequences rich in purines/pyrimidines) and thus form triple helixes.These oligonucleotides directed in this way against DNA have been called"anti-gene" or alternatively "anti-code". The formation of a triplehelix, at a particular sequence, can block the fixing of proteinsintervening in the expression of a gene and/or allow irreversible damageto be introduced into the DNA if the oligonucleotide under considerationpossesses a particular reactive group. Such antisense oligonucleotidescan form artificial restriction endonucleases, directed against specificsequences.

In cells, and more particularly in an organism, in the bloodcirculation, for example, the natural oligonucleotides are sensitive todegradation by nucleases. Nucleases are degradation enzymes capable ofcutting the phosphodiester bonds of DNA or of RNA, either by introducinginternal cleavages into mono- or bicatenary molecules, or by attackingthese molecules starting from their ends. The enzymes which attackinternally are called endonucleases and those attacking by the ends arecalled exonucleases.

The use of oligonucleotides encounters two major problems which are, onthe one hand, great sensitivity to degradation by exonucleases which arefound as well in the serum or in extracellular medium or inintracellular cytoplasmic medium and, on the other hand, lowintra-cellular penetration.

The use of modified oligonucleotides has already been proposed toincrease the stability of oligonucleotides or favor penetration throughcellular membranes or alternatively to stabilize hybridization andspecific affinity for a target sequence, whether this be a single ordouble strand nucleic acid, or even a protein, or alternatively toincrease the interaction with said target sequence. Chemicalmodifications of the structural skeleton of the molecule or derivationsor couplings to reactive or intercalating groups have been proposed, ingeneral localized at the end of the oligonucleotides.

As far as the chemical modifications of the skeleton are concerned, ithas been proposed to modify the nature of the internucleotide phosphatelinkage, especially in the form of methylphosphonate, phosphorothioateor phosphorodithioate; or alternatively by modifying the sugar part,especially by an alpha-anomeric configuration, a 2'--O--CH₃ substitutionor by replacing the oxygen of the furan ring by a sulfur(4'-thioribonucleotide). It has also been proposed to modify thenucleotide bases.

Thus, in French Patent Applications FR 83 01223 (2 540 122) and FR 8411795 (2 568 254) chemical compounds formed by an oligonucleotideincluding natural or modified linkage of β-nucleotides have beendescribed, on which are found, fixed by a covalent bond, at least oneintercalating group, which compounds have the property of selectivelyblocking the expression of a gene and which therefore are particularlyuseful in therapy as antiviral, antibiotic, antiparasitic or antitumorsubstances.

In International Application WO 88/04301, oligonucleotides ofalpha-anomeric configuration have been described having more stableparallel pairings with complementary sequences.

However, the uses proposed until now raise other problems, especially oftoxicity, as chemical modification introduced into the molecule prove tobe capable of inducing toxicity on the pharmacological level in certaintherapeutic applications. Generally speaking, it is difficult to combinethe different criteria of resistance to nucleases, stabilization ofhybridization, penetration into the cell and, likewise, increase inactivity yielding the duplex with the RNAse H substrate complementarytarget sequence, which has the property of cleaving the DNA/RNAduplexes.

The oligonucleotide compounds whose phosphate part was modified bymethylphosphonate are particularly resistent to degradation bynucleases. However, the electrically neutral oligomers such as theoligomers of the methylphosphonate type penetrate more easily into thecell than the electrically charged oligomers such as thephosphodiesters. However, these methylphosphonate derivatives have achirality, especially at the phosphate, thus leading to the formation ofdiastereoisomers. In addition, the RNA/oligodeoxynucleotidemethylphosphonate duplexes are not substrates of RNAseH. The oligomerderivatives of the phosphorothioate diester type have a resistance todegradation by nucleases but to a lesser degree than themethylphosphonate oligomers. On the other hand, they lead toelectrically charged oligomers capable of activating RNAse H, butpenetrate less easily into the cell than the methylphosphonateoligomers.

Generally speaking, the oligonucleotides which are the subject of theinvention are intended to provide novel stable oligonucleotides capableof being internalized in cells and capable of hybridizing and/or ofhaving an affinity for specific nucleic acid or protein sequences andthus of interacting with cellular or viral factors.

The present invention provides oligonucleotides with electricallyneutral phosphorothioate triester linkages being able to re-form, afterpenetration into the cell, and selectively, phophorothioate diester orpolar phosphodiester bonds. Analogously, the present invention includesphosphorodithioate triester linkages giving, intracellularly,phosphorodithioate diester or phosphorothioate diester linkages.

To do this, P--S⁻ bonds of an oligomer are selectively protected by abioreversible group (X) in the intracellular media.

The modifications proposed according to the invention produce oligomershaving the advantageous properties of phosphorothioate diesterderivatives, while overcoming the disadvantages of the latter,especially as far as their sensitivity to extracellular exonucleases andtheir difficulty in penetrating through the cellular membrane isconcerned.

The present invention thus relates more precisely to a phosphorothioateor phosphorodithioate triester oligonucleotide, characterized in that itcomprises internucleotide linkages which have a P--S⁻ bond protected bya bioreversible group (X) in intracellular media.

The route of synthesis used in this invention consists in the use of anucleophilic substitution reaction by the sulfur atom of the P--S⁻ bondof an alkylating agent XL, L being a leaving group (halogen, ester,tosyl . . . ) and X a bioreversible group. It follows that it ispossible to convert the P--S⁻ functions into corresponding bioreversiblephosphotriesters, as will be shown in the examples below.

Such a process does not require protection of heterocyclic bases and cantherefore be carried out directly on a previously preparedoligonucleotide. It is thus possible to obtain phosphorothioate triesteroligomers starting from phosphorothioate diester oligomers. In fact, amixed linkage comprising phosphodiester (P--O⁻) and phosphorothioate(P--S⁻) bonds will be selectively alkylated at the sulfur atoms.

The phosphorothioate triester oligomers according to the presentinvention are electrically neutral in the extracellular medium andtherefore benefit from improved penetration into the cell. In addition,they allow electrically charged phosphodiester or phosphorothioatediester oligonucleotides to be delivered intracellularly which arecapable, as such, of being substrates of RNAse H when they form a mixedRNA/DNA duplex with their target complementary sequences.

The principle of the invention applies to any synthetic oligonucleotide,of the DNA, RNA or mixed series, whatever the biological targetenvisaged, insofar as it has internucleotide linkages containing a P--S⁻bond capable of being alkylated by bioreversible groups.

In one embodiment, the oligonucleotides according to the presentinvention correspond to the formula ##STR1##

in which:

Y is O or S

R₁ and R₂ are respectively a residue in the 3'-O and

5'-O positions of a nucleoside or of an oligonucleotide theinternucleotide linkage of which is natural or modified, the linkage ofwhich is especially of the phosphorothioate triester type of formula##STR2## X is a --(CH₂)_(n) --Y¹ --W radical or X is a --(CH₂)_(n) --Y¹--W radical or

y¹ is S or O

n varies from 1 to 6

W is:

either SU with U being an optionally substituted alkyl, aryl or osideradical, or ##STR3## with y² being O or S, and Z being an optionallysubstituted alkyl, aryl or oside radical.

"Modified oligonucleotide" is understood here to mean modifications inthe sugar part, in the base or alternatively in the phosphate linkage,or even in the anomeric configuration of the linkages. These X groupsundergo an enzymatic cleavage of the Y₁ /W bond by the enzymaticactivation of intracellular enzymes according to a mechanism illustratedin FIG. 1.

When U and Z are an alkyl, aryl or oside radical, the C₁ and C₇ alkylradicals, benzyl and phenyl radicals and, as sugars, glucose, mannose orrhamnose are mentioned in particular.

Among these X groups, --(CH₂)_(n) --S--S--U or ##STR4## are moreparticularly mentioned and more particularly still ##STR5## where n=1 or2.

These phosphorothioate triester linkages are converted under theinfluence of intracellular enzymes either into phosphodiesters or intophosphorothioate diesters, as is shown by the decomposition studies oncellular extracts presented below and the mechanisms indicated in FIG.1.

Among the alkyls forming the groups U or Z, the lower alkyls optionallysubstituted by a group chosen particular.

Thus, in a particular embodiment of the invenvention, X is --(CH₂)_(n)--S--S--(CH₂) _(n).spsb.1 --X¹ with n and n¹ being an integer from 1 to4, preferably 2, and X^(l) is H, OH, SH or NH₂, or X is --(CH₂)_(n) --Y¹--CO--Z, where n=1 or 2 and Z=CH₃ or tBu. More particularly still X =tBuCOOCH₂ --, CH₃ COSHCH₂ CH₂ --or CH₃ COSCH₂ --.

The electrically charged phosphorothioate diester oligomers are somewhatinsensitive to intracellular nuclease degradation. This is why, in anadvantageous embodiment according to the invention, the oligonucleotidestides will be formed by a chimeric oligomer comprising a central DNA orRNA sequence, the internucleotide linkages of which contain a ##STR6##bond, this central sequence being flanked in the 5' and 3' positions byDNA or RNA sequences modified so that they are resistant to nucleasesand/or stabilize hybridization with a complementary strand.

This embodiment is particularly advantageous in the case of ananti-sense approach in which an oligonucleotides nucleotide of the DNAtype is used so as to form a DNA/RNA duplex which will thus be asubstrate of RNAse H. These chimeric oligomers according to theinvention combine all the advantageous properties of cellularpenetration, resistance to intra- and extracellular enzymes andformation of duplex substrates of RNAse H.

In particular, compounds of formula I will be used in which R₁, and R₂have sequences at the 5' and 3' ends with electrically neutralinternucleotide linkages which are resistant to degradation by extra-and intra-cellular nucleases, such as linkages of the methyl-phosphonatetype.

According to another embodiment, the oligonucleotide according to theinvention are formed from a chimeric oligomer comprising a β-anomericcentral DNA or RNA sequence, the internucleotide linkages of which areof the type Y=P-SX, this central sequence being flanked in the 5' and 3'positions by alpha-anomeric DNA or RNA sequences.

In particular, an oligonucleotide according to the invention can beformed from a chimeric oligomer containing a central sequence ofphosphorothioate triester β-nucleoside linkage with internucleotidelinkage of the ##STR7## type surrounded by flanks formed from sequencesof phosphate diester form alpha-nucleoside linkages.

Generally speaking, different nucleotides can be part of the formulationof oligonucleotides according to the invention. The oligonucleotides towhich the present invention relates can be formed by a sequence ofnucleotide bases including adenine (A), guanine (G), cytosine (C),thymine (T) and uracil (U), or the oligonucleotides can likewise includerare nucleotides (inosine, I, or rI, for example) or modifiednucleotides, either in the deoxyribo- series or in the ribo- series,interconnected by non-modified or modified phosphodiester bondsaccording to the present invention.

In particular, the oligonucleotides can include reactive nucleotidescapable of establishing bonds with the sequence of the target moleculecomplementary to the oligonucleotide.

Thus, the oligonucleotides according to the invention can carry reactivegroups grafted onto the nucleotides, such as, for example psoralengroups, or other bridging agents or intercalating agents able to reactwith the sequence of the target molecule complementary to theoligonucleotide.

Likewise oligonucleotides form part of the invention, which are coupledto molecules allowing their intracellular penetration to be increased,and in particular lipophilic groups, polypeptides or proteins.

The oligonucleotides according to the invention are prepared startingfrom an oligonucleotide having phosphothioate diester internucleotidelinkages of the type ##STR8## A nucleophilic substitution reaction bythe sulfur atom of the --P--S⁻ bond of said phosphorothioate diesterlinkages is carried out on an alkylating agent XL, thus leading to theformation of an oligonucleotide with phosphorothioate triester linkages##STR9## L being a leaving group such as halogen, ester or tosyl, and Xbeing a bioreversible group according to the invention.

The phosphorothioate diester oligonucleotides with a ##STR10## linkageare prepared by conventional methods of synthesis, by a chemical orbiochemical route, or by approaches calling for combinations of chemicalsynthesis and molecular biology techniques.

Among the methods described as conventional, various methods of chemicalsynthesis of oligonucleotides have been developed and are well known tothe specialists working in these fields. For example, one methodconsists in using a solid support called CPG (controlled pore glass) towhich the first nucleoside is fixed covalently by a spacer through its3' OH end. The 5' OH end of the nucleoside is protected by an acid-labeldi-p-methoxytrityl group. This approach, using phosphite triesterchemistry, and in which deoxynucleoside 3'-phosphoramidite are used assynthons is called the phosphoramidite method. This approach is mostused at present and has the advantage of being entirely automatic.

Another approach used for the synthesis of oligonucleotides is that ofphosphonate chemistry. This approach starts with the condensation of adeoxynucleoside 3'-H-phosphonate with a deoxynucleoside coupled to aglass or silica support. Successive condensation cycles lead to thesynthesis of oligonucleotide H-phosphonates. These oligonucleotides areoxidized in one step to give the phosphodiesters.

Using one or the other of these techniques, or any other sequentialprocedure allowing the synthesis of polynucleotide chains of determinedsequence in advance, oligonucleotides having the desired startingstructure are obtained.

Detailed syntheses of phosphorothioate diester oligonucleotides havebeen described, for example, in J. Am. Chem. Soc. 106: 6077-6079 (1984)and Nucleic Acids Res. 14: 5399-5407 (1986).

The oligonucleotides can be used as various diagnostic, cosmetic orpharmacological compositions at variable concentrations and with theappropriate excipients according to the applications.

Other characteristics and advantages of the present invention willappear in the light of the examples which follow.

FIG. 1 shows the mechanisms of intracellular decomposition ofbioreversible groups.

I--Examples 1 to 3: Synthesis of phosphorothioate triester dinucleosides4a, 4b, and 4c and evaluation of their stability in biological medium

I-1--Synthesis of the compounds 4a, 4b and 4c

General conditions

Thin-layer chromatography (TLC) is carried out on Merck 60 F₂₅₄ silicagel sheets. The products are detected with a UV lamp (254 nm) andvisualized by heating after spraying with 10% sulfuric acid in 95°ethanol. Chromatography on a silica gel column is carried out withsilica Kieselgel 60 (40 μm-63 μm).

Proton NMR spectra are recorded at ambient temperature on a Bruker AC250 apparatus. The samples are solubilized in DMSO-d₆. The chemicalshifts (δ) are expressed in ppm with reference to the signal of DMSO-d₅fixed at 2.49 ppm, taken as internal reference. The coupling constantsare given in Hertz. The multiplicity of the signals observed isindicated by a small letter:

m: multiplet; pq: pseudo-quartet, t: triplet; d: doublet; s: singlet;sl: broad singlet The nucleoside having its 5' OH function free isrepresented sented by N₁ and the nucleoside having its 3' OH functionfree is represented by N₂.

The phosphorus NMR spectra are recorded at ambient temperature on aBruker WP 200 SY apparatus with proton decoupling. The samples aresolubilized in CD₃ CN or in DMSO-d₆. The chemical shifts are expressedin ppm in relation to 66% H₃ PO₄ taken as external reference.

The mass spectra are carried out on a JEOL JMS DX 300 apparatus by theFAB ionization method in positive mode with different matrices: glycerol(G), glycerol/-thioglycerol (GT) or 3-nitrobenzyl alcohol (NBA).

The acetonitrile was distilled after heating to reflux for one nightover calcium hydride and is kept over molecular sieve (4A).

The alkylating agents were synthesized according to processes describedin the literature:

pivaloyloxymethyl iodide: European Patent Appl. No. 843080672

bromoethylacetyl sulfide: P. Nylen, A. Olsen, Svensk Kem. Tid., 53, 274(1941)

bromomethylacetyl sulfide: G. K. Farrington, A. Kumar, F. C. Wedler,Oppi briefs, 21, 390 (1989).

The phosphorothioate diester dinucleosides were synthesized according toconventional methods described in the literature, using H-phosphonatechemistry (J. Stawinski, M. Thelin, Nucleosides and Nucleotides, 9, 129(1990), P.J. Garegg, T. Regberg, J. Stawinsky, R. Stromberg, Nucleosidesand Nucleotides, 6, 655 (1987)).

Aqueous solutions of 1M and 0.5M triethylammonium hydrogen carbonate(TEAB) were used to neutralize the reaction media and to carry out theextractions.

Before lyophilization, the solutions are filtered on a Millex HV-4filter (Millipore, 0.45 μm).

Synthesis: ##STR11##

EXAMPLE 1 O- 5'-O-(4,4'-Dimethoxytrityl) thymidin-3'yl! O-3'-O-(4,4'-dimethoxytrityl)thymidin-5'-yl! S-(pivaloyloxymethyl)phosphorothioate (3a)

Pivaloyloxymethyl iodide (2a) (484 mg, 2 mmol) is added to a solution ofphosphorothioate diester dinucleoside (1) (254 mg; 0.2 mmol) inanhydrous acetonitrile (10 ml). The reaction is stirred at ambienttemperature for 30 minutes, then the reaction medium is neutralized byaddition of an aqueous solution of 1M TEAB (2 ml) and left for 5 minuteswith stirring. The reaction mixture is then poured onto an aqueoussolution of 0.5M TEAB (50 ml) and extracted with dichloromethane (3×30ml). The organic phases are collected, washed with water (50 ml), driedover sodium sulfate, filtered and then evaporated to dryness. Theresidue is then chromatographed graphed on a silica gel column using agradient of methanol (0 to 2%) in dichloromethane as eluent. Thefractions containing (3a) are combined and concentrated under reducedpressure, leading to a white solid residue (220 mg; 86%) which iscarefully dried for use in the following step.

R_(f) =0.67 (CH₂ Cl₂ /MeOH: 9/1).

O-(Thymidin-3'-yl) O-(thymidin-5'-yl) S-(pivaloyloxymethyl)phosphorothioate (4a)

The totally protected dimeric phosphorothioate triester (3a) isdissolved in a mixture of acetic acid/water/methanol (8:1:1, v:v:v) (10ml). The reaction is left with stirring at ambient temperature for 5hours. The reaction medium is then evaporated and the residue iscoevaporated several times with water and toluene. The residue is thenchromatographed on a silica gel column using a gradient of methanol (0to 10%) in dichloro-methane as eluent. The fractions containing theproduct (4a) are collected, concentrated under reduced pressure, andthen the residue is coevaporated several times with dioxane andlyophilized in dioxane. (4a) is obtained in the form of a white powder(98 mg, 85%).

R_(f) =0.2 (CH₂ Cl₂ /MeOH: 9/1); MS: FAB>0 (NBA): 677: (M+H)⁺ ; 551:(M-B)⁺ ; 424: (M- (2B+2H))⁺ ; ³¹ P NMR (CD₃ CN): δ=27.21 and 27.31 ppm(2 diastereoisomers); ¹ H NMR (DMSO-d₆): δ=11.34 (sl. 2H, 2NH); 7.68 and7.48 (2m, 2H, 2H₆): 6.21 (m, 2H, 2H_(1')); 5.48 (d, 1H, OH_(3'), J=4.4Hz); 5.41 (d, 2H, SCH₂ O, J=20 Hz); 5.26 (t, 1H, OH_(5'), J=5.1 Hz),5.10 (m, 1H, H_(3') (N₁)); 4.26 (m, 3H, H_(3') (N₂), H_(5') and H_(5")(N₂)); 4.12 (m, 1H, H_(4') (N₁)); 3.96 (m, 1H, H_(4') (N₂)); 3.61 (m,2H, H_(5') and H_(5") (N₁)); 2.38 (m, 2H, H_(2') and H_(2") (N₁)); 2.14(m, 2H, H_(2') and H_(2") (N₂)); 1.79 and 1.77 (2s, 2CH₃ (N₁ and N₂));1.13 (d, 9H, (CH₃)₃ C) ppm.

EXAMPLE 2 O- 5'-O-(4,4'-Dimethoxytrityl)thymidin-3'-yl!O- 3'-O-(4,4'-dimethoxytrityl) thymidin-5'-yl! S- (acetylthioethyl)phosphorothioate (3b)

Bromoethylacetyl sulfide (2b) (360 mg; 1.97 mmol) is added to a solutionof phosphorothioate diester dinucleoside (1) (50 mg; 0.0394 mmol) inanhydrous acetonitrile (5 ml). The reaction mixture is stirred atambient temperature for 5 days, then it is neutralized by addition of anaqueous solution of 1M TEAB (1 ml) and left with stirrring for 5minutes. The reaction mixture is then poured onto an aqueous solution of0.5M TEAB (25 ml) and extracted with dichloromethane (3×15 ml). Theorganic phases are collected, washed with water (25 ml), dried oversodium sulfate, filtered and then evaporated to dryness. The residue isthen chromatographed graphed on a column of silica gel using a gradientof methanol (0 to 4%) in dichloromethane as eluent. (3b) is obtained inthe form of a white solid (27 mg, 55%).

R_(f) =0.75 (CH₂ Cl₂ /MeOH: 9/1).

O- (Thymidin-3'-yl) O- (thymidin-5'-yl) S- (acetylthioethyl)phosphorothioate (4b)

The totally protected phosphorothioate triester dinucleoside (3b) isdissolved in an acetic acid/water/ methanol mixture (8:1:1, v:v:v) (5ml) and left with stirring all night. The following day, the reactionmixture is evaporated and the residue is coevaporated several times withwater and then with toluene. The solid residue is then chromatographedon a column of silica gel using a gradient of methanol (0 to 10%) indichloro methane as eluent. The fractions containing the detritylatedphosphorothioate triester dinucleoside (4b) are collected and evaporatedunder reduced pressure, then the residue is coevaporated with dioxaneand lyophilized in dioxane. (4b) is obtained in the form of a whitepowder (12 mg, 86%).

R_(f) =0.35 (CH₂ Cl₂ /MeOH: 9/1); MS: FAB>0 (GT): 665: (M+H)⁺ ; ³¹ P NMR(DMSO-d₆): δ=28.09 and 28.22 ppm; ¹ H NMR (DMSO-d₆): δ=11.34 (2s, 2H,2NH); 7.68 and 7.48 (2d, 2H, 2H₆); 6.20 (t, 2H, 2H_(1')); 5.48 (d, 1H,OH_(3')), 5.26 (pq, 1H, OH_(5')), 5.09 (m, 1H, H_(3') (N₁)), 4.26 (m,3H, H_(3') (N₂), H_(5') and H_(5") (N2)); 4.10 (m, 1H, H_(4') (N₁));3.95 (m, 1H, H_(4') (N₂)); 3.61 (m, 2H, H_(5') and H_(5") (N₁)); 3.13and 3.02 (2m, 4H, SCH₂ CH₂ S); 2.29 (m, 2H, H_(2') and H_(2") (N₁));2.33 (d, 3H, CH₃ CO); 2.13 (m, 2H, H_(2') and H_(2") (N₂)); 1.77 and1.78 (2s, 6H, 2CH₃ (N₁ and N₂)) ppm.

EXAMPLE 3 O- 5'-O-(4,4'-Dimethoxytrityl)thymidin-3'-yl! O-3'-O-(4,4'-dimethoxytrityl) thymidin-5'-yl! S-(acetylthiomethyl)phosphorothioate (3c)

Iodomethylacetyl sulfide (98 mg; 0.457 mmol) is added to a solution ofphosphorothioate diester dinucleoside (1) (58 mg; 0.0457 mmol) inanhydrous acetonitrile (5 ml). The reaction is left with stirring allnight at ambient temperature, then the reaction mixture is neutralizedby addition of a 1M aqueous solution of TEAB (1 ml) and left withstirring for several minutes. The reaction mixture is then poured onto a0.5M aqueous solution of TEAB (25 ml) and extracted with dichloromethane(3×15 ml). The organic phases are collected, washed with water (25 ml),dried over sodium sulfate, filtered and then evaporated to dryness. Theresidue is then chromatographed on a column of silica gel using agradient of methanol (0 to 3%) in dichloromethane as eluent. (3c) isobtained in the form of a white solid (46 mg, 80%).

R_(f) =0.55 (CH₂ Cl₂ /MeOH: 9/1).

O-(Thymidin-3'-yl) O-(thymidin-5'-yl) S-(acetylthiomethyl)phosphorothioate (4c)

The totally protected phosphorothioate dinucleoside (3c) is dissolved inan acetic acid/water/methanol mixture (8:1:1, v:v:v) (5 ml) and leftwith stirring at ambient temperature all night. The following day, thereaction mixture is evaporated and coevaporated several times with waterand with toluene. The residue is then chromatographed on a column ofsilica gel using a gradient of methanol (0 to 10%) as eluent. Thefractions containing the detritylated phosphorothioate triesterdinucleoside (4c) are collected and concentrated under reduced pressure.The residue is coevaporated with dioxane and then lyophilized indioxane. (4c) is obtained in the form of a white powder (20 mg, 85%).

R_(f) =0.62 (CH₂ Cl₂ /MeOH: 8/2); MS: FAB>0 (GT): 651 (M+H)⁺ ; ³¹ P NMR(CD₃ CN): δ=27.22 and 27.44 ppm (2 diastereoisomers); ¹ H NMR (DMSO-d₆):δ=11.34 (d, 2H, 2 NH); 7.70 and 7.49 (2d, 2H, 2H₆); 6.22 (m, 2H,2H_(1')); 5.48 (d, 1H, OH_(3')); 5.37 (m, 1H, OH_(5')); 5.08 (m,1H,H_(3') (N₁)); 4.29 (m, 5H, H_(3') (N₂), H_(5') and H_(5") (N₂), SCH₂ S);4.13 (m, 1H, H_(4') (N₁)); 3.97 (m, 1H, H_(4') (N₂)); 3.62 (m, 2H,H_(5') and H_(5') (N₁)), 2.39 (m, 5H, H_(2') and H_(2") (N₁), CH₃ CO),2.15 (m, 2H, H_(2') and H_(2") (N₂)); 1.78 (s, 6H, 2CH₃ (N₁ and N₂)).

I-2--Stability studies on dimeric phosphorothioate triesters 4a, 4b and4c General conditions

Method

The stability of the dimers (4a), (4b) and (4c) in different biologicalmedia were studied according to an HPLC technique perfected in thelaboratory (A. Pompon, I. Lefebvre, J. L. Imbach, BiochemicalPharmacology 43, 1769 (1992)), which does not require any preliminaryhandling of the sample and allows its direct injection; a precolumn,which allows proteins to be eliminated, filled with a new material ISRP(Internal Surface Reverse Phase) is combined with a high resolutioncolumn (inverse phase) allowing chromatographic analysis.

Equipment

The chromatograph (Waters-Millipore) is composed of:

an M 680 programmer

two M 510 pumps

a WISP 712 automatic injector

an M 990 UV diode array detector

an NEC APC IV microcomputer

a Waters 990 printer

a model 7010 6-way valve (Rheodyne).

The columns are supplied by SFCC Shandon:

ISRP precolumn (Ultrabiosep C₁₈, 10 μm, 4.6 mm×10 mm)

Inverse phase analytical column (Nucleosil C18, 5 μm, 4.6 mm×100 mm) Theanalytical column is thermostatted at 30° C.

The analysis of the results is carried out on the EUREKA software.

Chemical products

Distilled water is purified on a MilliQ system (Waters-Millipore),Acetonitrile is of HPLC-far UV quality (Fisons), Ammonium acetate is of"analytical" quality (MERCK), The culture media are composed of 90% ofRPMI 1640 and 10% of heat-inactivated fetal calf serum (GIBCO), Thecellular extracts were kindly supplied by Miss A. -M. Aubertin(University of Strasbourg I). They are prepared in the following way:CEM cells in the exponential growth phase are separated from the culturemedium by centrifugation (10⁴ g, 4 min, 40°). The residue (approximately100 μl, 5×10⁷ cells) is dissolved in 2 ml of buffer (Tris-HCl 140 mM,pH=7.4) and sonicated. The lysate is centrifuged (10⁵ g, 1 h, 4°) toeliminate membranes, organelles and chromatin. Two types of eluents wereutilized for the HPLC:

eluent A: 0.1M ammonium acetate buffer, pH=5.9,

eluent B: 0.1M ammonium acetate buffer, 50% acetonitrile, pH=5.9.

Preparation of the samples

A 10⁻² M solution of the compound to be studied is prepared in DMSO.

This solution is diluted with water to give a parent solution ofconcentration 5×10⁻⁴ M.

a) Study of stability in culture medium:

100 μl of parent solution of the compound to be studied are added to 900μl of culture medium which has previously been filtered on a Millex GVsterile filter (Millipore, 0.22 μm). After mixing, fractions (100 μl)are distributed in sterile Eppendorf tubes. These tubes are placed in anoven at 37° C. and removed as a function of the kinetic development. Thesamples are immediately analyzed by HPLC (volume injected: 80 μl) orpreserved at -25° C. with a view to subsequent analysis.

b) Study of stability in cellular extract: 100 μl of parent solution ofthe compound to be studied are added to 900 μl of cellular extract whichhas previously been filtered on a Millex GV sterile filter (Millipore,0.22 μm). After mixing, fractions (100 μl) are distributed in Eppendorftubes. These tubes are placed in an oven at 37° C. and removed as afunction of the kinetic development. The samples are immediatelyanalyzed by HPLC.

Results

The results regarding the stability studies of the three dimers (4a),(4b) and (4c) are collected in the table below:

C₀ =5×10⁻⁵ M

t_(l/2) =time at the end of which half of the starting product hasdecomposed

PO⁻ =phosphodiester dinucleoside

PS⁻ =phosphorothioate diester dinucleoside

% PO⁻ , % PS⁻ =molar fractions of phosphodiester dinucleoside and ofphosphorothioate diester dinucleoside, expressed in relation to theinitial quantity of phosphorothioate triester dinucleoside.

The percentage of PS⁻ corresponds to the quantity of phosphorothioatediester dinucleoside formed after complete decomposition of the startingphosphorothioate triester dinucleoside (considering that thephosphorothioate diester dinucleoside is stable under the condition usedand accumulates).

The percentage of PO⁻ is calculated by the difference (100-% PS⁻ formed)because the phosphodiester dinucleoside decomposes rapidly from itsformation.

    ______________________________________                                        RPMI + 10% serum cellular extract                                             t.sub.1/2                                                                              % PO.sup.-                                                                            % PS.sup.-                                                                            t.sub.1/2 % PO.sup.-                                                                          % PS.sup.-                           ______________________________________                                        (4a)  6 h    80      20    40    min   /     100                              (4b)  7 h    50      50    10    min   /     100                              (4c)  1 h    20      80    5-10  min   /     100                              ______________________________________                                    

It follows from the stability study of the compounds 4a-c presentedabove that these are rapidly and selectively converted in cellularextract into their corresponding phosphorothioate diester, which is inagreement with the original hypothesis.

II--Examples 4 to 6: Synthesis of oligonucleotides containingphosphorothioate triester bonds by postsynthesis alkylation ofphosphorothioate diester bonds. Evaluation of their stability inbiological media

II-1--Synthesis of oligonucleotides 5c, 6b and 7b containingphosphorothioate triester bonds

General conditions

The oligonucleotides containing phosphorothioate diester bonds weresynthesized on a solid phase in an Applied Biosystems model 381 A DNAsynthesizer, on support columns corresponding to one μmole of graftednucleoside.

The purification and analysis of the oligonucleotides nucleotides 5c, 6band 7b were carried out by HPLC using a chromatographic system composedof Waters-Millipore equipment:

an M 680 programmer

an NEC APC IV computer

a Waters 990 printer

two M 510 pumps

a U₆ K injector

a UV diode array detector

The columns and the precolumns are supplied by SFCC-Shandon:

in the case of preparative HPLC, a Nucleosil C₁₈ N 525 column (10 mm×250mm) of particle size 5 μm was used, protected by a Nucleosil C₁₈ PSF-25precolumn of particle size 5 μm,

in the case of analytical HPLC, a Nucleosil C₁₈ N 125 column (4.6 mm×150mm) of particle size 5μm protected by a Nucleosil C₁₈ PSF-25 precolumnof particle size 5 μm used.

After purification, the oligonucleotides are filtered on a Millex HV-13filter (Millipore, 0.45 μm) and submitted to several successivelyophilizations in water.

Following alkylation reactions of the oligonucleotides and ofpurification of the alkylated oligonucleotides 5c, 6b and 7b werecarried out by HPLC on a Waters-Millipore chromatograph composed of:

a 600 E programmer

a 600 pumping system

a U₆ K manual injector

a 486 UV detector

a Powermate SX Plus microcomputer.

Follwing of alkylation reactions was carried out on a Nucleosil C₁₈ 5 μmcolumn (4.6 mm×150 mm) protected by a Nucleosil C₁₈ 5 μm precolumn(SFCC - Shandon).

The purification of the oligonucleotides 5c, 6b and 7b was carried outon a RADIAL-PAK C₁₈ 10 μm cartridge to support the RCM column (8×10),protected by a GUARD-PAK Resolve C₁₈ precolumn (Waters-Millipore).

The desalting of the samples was carried out on a C₁₈ SEP--PAK cartridge(Waters-Millipore).

Before lyophilization, the solutions are filtered on a Millex HV-13filter (Millipore, 0.45 μm).

II-1-1--EXAMPLE 4

Synthesis of the chimeric dodecamer 5c containing a phosphorothioatetriester central window and methylphosphonate flanks

a)--Synthesis of a dodecamer containing a phosphorothioate diestercentral window and methylphosphonate flanks ##STR12##

The standard elongation cycle proposed for the synthesis ofoligonucleotides was used, using the methylphosphonamidite synthon forthe flanks and the mixture I₂ /THF/water/pyridine as oxidation agent,and the cyanoethyl phosphoramidite synthon and the Beaucage reagent assulfuration agent (R. P. Iyer, W. Egan, J. B. Regan, S. L. Beaucage, J.Am. Chem. Soc., 112, 1253 (1990)) for the central part (3 incorporationcycles).

The detachment of the oligonucleotide from the support and itsdeprotection were carried out according to the procedure conventionallydescribed for methylphosphonates (P. S. Miller, M. P. Reddy, A.Murakami, K. R. Blake, S. B. Lin, C. H. Agris, Biochemistry, 25, 5092(1986)) using a solution of ethylenediamine in absolute ethanol (1:1,v:v).

The synthesis on the one micromole scale led us to a rough synthesisfigure of 75 absorption units at 260 nm (A₂₆₀ units).

After purification by preparative HPLC, we obtained 37 A₂₆₀ units ofchimeric oligonucleotide of a spectrophotometric purity of 100%(determined by HPLC analysis).

b)--Alkylation of the phosphorothioate diester bonds. ##STR13##

The following are introduced successively into an Eppendorf:

120 μl of acetonitrile

135 μl of 20 mM phosphate buffer (pH=6.15)

15 μl of iodomethylacetyl sulfide in 0.92M solution in acetonitrile

30 μl of 7.66 mM solution of oligonucleotide in water (beingapproximately 22 A26 0 units)

The Eppendorf is placed in a dry bath previously thermostatted at 50° C.

Samples of 5 μl are taken at different reaction times and are analyzedby HPLC in order to estimate the progress of the reaction.

After 16 hours of reaction, the presence of the starting oligonucleotideis no longer detected (t_(r) =12.99 min) and the totally alkylatedoligonucleotide (t_(r) =17.53 min) is purified by HPLC.

The oligonucleotide is then desalted, before being lyophilized in awater/dioxane mixture (50:50, v:v) and analyzed again by HPLC.

10 A₂₆₀ units of totally alkylated oligonucleotide of aspectrophotometric purity of 97% were obtained (determined by HPLC).

II-1-2--- EXAMPLE 5

Synthesis of the entirely phosphorothioate thioate triester dodecamer 6b

a)--Synthesis of a phosphorothioate diester dodecamer d(C_(P)(S)A_(p)(s) C_(p)(s) C_(p)(s) C_(p)(s) A_(p)(s) A_(p)(s) T_(p)(s) T_(p)(s)C_(p)(s) T_(p)(s) G) SEQ. ID. NO: 1,

The standard cycle of elongation proposed for the synthesis ofoligonucleotides using cyanoethyl phosphoramidite synthons of the fourbases suitably protected and replacing the oxidation step with I₂/THF/water/pyridine by a sulfuration step with the Beaucage reagent wasused.

The detachment of the oligonucleotide from the support and itsdeprotection were carried out by treatment with concentrated ammonia(32%) overnight at 55° C.

b)--Alkylation of the phosphorothioate diester bonds

Phosphate buffer (pH=6.15, 123 μl), acetonitrile (120 μl) and a 1Msolution of iodoethylacetyl sulfide (2d) (55 μl) are added successivelyto a solution of 6.4 mM phosphorothioate diester oligonucleotide,d(C_(P)(S) A_(p)(s) C_(p)(s) C_(p)(s) C_(p)(s) A_(p)(s) A_(p)(s)T_(p)(s) T_(p)(s) C_(p)(s) T_(p)(s) G) SEQ. ID. NO: 1, in water (42 μl).The mixture is maintained at 50° C. for 48 hours. After evaporation, theresidue is purified by HPLC. The fraction containing the completelyalkylated phosphorothioate dodecamer 6b is desalted and the evaporationresidue taken up again in a water/dioxane mixture (50:50, v:v, 1 ml) islyophilized.

Yield 35%. Reference (2d) refers to the nomenclature used in thesynthesis scheme of the paragraph I-1.

II-1-3--EXAMPLE 6

Synthesis of the chimeric dodecamer 7b containing a central windowformed of phosphorothioate triester β-nucleosides and of flanks formedof phosphate diester α-nucleosides.

a)--Synthesis of the dodecamer containing a β-phosphorothioate diesterwindow and of d α-(TCTT)3'→3'β-(T_(P)(s) T_(p)(s) C_(p)(s) C)5'→5'α-(CTCT)! SEQ. ID. NO: 2α-phosphate diester flanks

The standard cycle of elongation proposed for the synthesis ofoligonucleotides using α-anomeric cyanoethyl phosphoramidite synthonssuitably protected on the bases for the flanks and the mixture I₂/THF/water/pyridine as oxidizing agent, and the β-anomeric cyanoethylphosphoramidite synthons suitably protected on the bases for the centralpart and the Beaucage reagent as sulfuration agent were used.

b)--Alkylation of the phosphorothioate diester bonds

Phosphate buffer (pH=6.15, 145 μl) and a solution of 1M iodoethylacetylsulfide (2d) (55 μl) are added successively to an 11 mM solution of dα-(TCTT)3'→3'β-(T_(P)(s) T_(p)(s) C_(p)(s) C)5'→5'α-(CTCT)! SEQ. ID. NO:2oligonucleotide in water (20 μl). The mixture is maintained at 50° C.for 48 hours. After evaporation, the residue is purified by HPLC. Thefraction containing the dodecanucleotide (7d) alkylated on the threecentral phosphorothioate bonds is desalted and then lyophilized. Yield47%.

II-2--Study of stability of the chimeric oligonucleotides nucleotides5c, 6b and 7b in biological media.

General conditions

The general conditions are the same as those used for the study ofstability of the phosphorothioate triester dimers, as far as the method,the equipment and the chemical products are concerned.

Preparation of the samples

A parent solution of oligonucleotide is prepared in dioxane (10 A₂₆₀units of oligonucleotide in 200 μl of dioxane).

a)--Study of stability in culture medium:

30 μl of parent solution of oligonucleotide (being approximately 1.5A₂₆₀ units) are removed, which are added to 1470 μl of culture mediumwhich has previously been filtered on a sterile Millex GV filter(Millipore 0.22 μm). After mixing, fractions (100 μl) are distributed insterile Eppendorf tubes. These tubes are placed in an oven at 37° C. andremoved as a function of the kinetic development. The samples areimmediately analyzed by HPLC (volume injected 80 μl) or preserved at-25° C. with a view to subsequent analysis.

b)--Study of stability in cellular extract:

10 μl of oligonucleotide parent solution are added to 990 μl of cellularextract which have previously been filtered on a sterile Millex GVfilter (Millipore, 0.22 μm). After mixing, fractions (100 μl) aredistributed in sterile Eppendorf tubes, placed in an oven at 37° C.,sampled at different times and immediately analyzed by HPLC.

Results

The results regarding the stability studies of the threeoligonucleotides 5c, 6b and 7b are collected in the table below.

    ______________________________________                                               RPMI + 10% serum  cellular extract                                              t.sub.1/2 of dis-                                                                          t.sub.1/2 of dis-                                                                        t.sub.1/2 of formation                       oligo-   appearance of                                                                              appearance of                                                                            of the totally                               nucleotide                                                                             the starting the starting                                                                             deprotected                                  studied  oligonucleotide                                                                            oligonucleotide                                                                          oligonucleotide                              ______________________________________                                        5c       40 min       <5 min     20 min                                       6b       55 min       <2 min     25 min                                       7b       35 min       <2 min     20 min                                       ______________________________________                                    

All of the data regarding the compounds 5c, 6b and 7b shows withoutambiguity that it is possible to protect internucleotidephosphorothioate functions selectively by bioreversible groups. Inaddition, it is indeed confirmed in cellular extracts that suchphosphorothioate triester oligonucleotides are rapidly deprotected.

FIG. 1 shows the mechanism of decomposition of bioreversible groupsaccording to the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CACCCAATTCTG12                                                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TCTTTTCCCTCT12                                                                __________________________________________________________________________

We claim:
 1. An oligonucleotide comprising the general formula ##STR14##in which: Y is O or S;R₁ and R₂ are, respectively, a residue in the 3'-O and 5' -O positions of a nucleoside or of an oligonucleotide, theinternucleotide linkage of which is natural or modified; X is--(CH₂)_(n) --S--S--U, --(CH₂)_(n) --O--C (═Y²)--Z or --(CH₂)_(n)--S--C(═O) --Z; each n is independently an integer from 1 to 6; U is anoptionally substituted alkyl, aryl or sugar residue; Y² is O or S; and Zis optionally substituted alkyl or sugar residue.
 2. An oligonucleotideaccording to claim 1, where n=1 or
 2. 3. An oligonucleotide comprisingthe general formula ##STR15## in which: Y is O or S;R₁ and R₂ arerespectively a residue in the 3' -O and 5' -O positions of a nucleosideor of an oligonucleotide, the internucleotide linkage of which isnatural or modified; X is a --(CH₂)_(n) --Y¹ --W radical, where y¹ is Sor O; each n is independently an integer from 1 to 6; and W is either SUor --C(Y²)--Z, where U and Z are a lower alkyl, optionally substitutedby one or more groups selected from the group consisting of OH, SH andNH₂, and where Y² is O or S.
 4. An oligonucleotide according to claim 1,characterized in that U and Z are a lower alkyl, optionally substitutedby one or more groups selected from the group consisting of OH, SH andNH₂.
 5. An oligonucleotide according to claim 3, characterized in that Xis --(CH₂)_(n) --S--S--(CH₂)_(n1) --X¹, wherein n and n¹ are an integerfrom 1 to 4, and X¹ is H, OH, SH or NH₂, or X is --(CH₂) _(n) --Y¹ --C(═O)--Z, where Z=CH₃ or tBu.
 6. An oligonucleotide according to claim 1,characterized in that it is formed of a chimeric oligomer comprising acentral DNA or RNA sequence, the internucleotide linkages of whichcomprise a ##STR16## bond, this central sequence being flanked in the 5'and 3' positions by DNA or RNA sequences modified so that they areresistant to nucleases and/or stabilize hybridization with acomplementary strand.
 7. An oligonucleotide according to claim 1,wherein said internucleotide linkage is a phosphorothioate triester. 8.An oligonucleotide according to claim 3, characterized in that it isformed of a chimeric oligomer comprising a central DNA or RNA sequence,the internucleotide linkages of which comprise a ##STR17## bond, thiscentral sequence being flanked in the 5' and 3' positions by DNA or RNAsequences modified so that they are resistant to nucleases and/orstabilize hybridization with a complementary strand.
 9. Anoligonucleotide according to claim 5, characterized in that it is formedof a chimeric oligomer comprising a central DNA or RNA sequence, theinternucleotide linkages of which comprise a ##STR18## bond, thiscentral sequence being flanked in the 5' and 3' positions by DNA or RNAsequences modified so that they are resistant to nucleases and/orstabilize hybridization with a complementary strand.
 10. Anoligonucleotide comprising the general formula ##STR19## in which: Y isO or S;R₁ and R₂ are respectively a residue in the 3' -O and 5' -Opositions of a nucleoside or of an oligonucleotide, the internucleotidelinkage of which is natural or modified; X is a --(CH₂)_(n) --Y¹ --Wradical whereY¹ is S or O; each n is independently an integer from 1 to6; and W is either SU or --C(Y²)--Z, whereU is an optionally substitutedalkyl, aryl or sugar residue; Y² O or S; and Z is an optionallysubstituted alkyl, aryl or sugar residue; wherein R₁ and R₂ haveelectrically neutral sequences at the 5' and 3' ends respectively, whichare resistant to degradation by exonucleases; and wherein theinternucleotide linkages at the 5' and 3' ends respectively of R₁ and R₂are of the methylphosphonate type.
 11. An oligonucleotide comprising thegeneral formula ##STR20## in which: Y is O or S;R₁ and R₂ arerespectively a residue in the 3' -O and 5' -O positions of a nucleosideor of an oligonucleotide, the internucleotide linkage of which isnatural or modified; X is a --(CH₂)_(n) --Y¹ --W radical whereY¹ is S orO; each n is independently an integer from 1 to 6; and W is either SU or--C(Y²)--Z, whereU is an optionally substituted alkyl, aryl or sugarresidue; Y² is O or S; and Z is an optionally substituted alkyl, aryl orsugar residue; wherein said oligonucleotide is formed from a chimericoligomer comprising a β-anomeric central DNA or RNA sequence, theinternucleotide linkages of which are of the ##STR21## type, thiscentral sequence being flanked in the 5' and 3' positions by α-anomericDNA or RNA.
 12. An oligonucleotide according to claim 1, characterizedin that said oligonucleotide is formed from a chimeric oligomercomprising a β-anomeric central DNA or RNA sequence, the internucleotidelinkages of which are of the ##STR22## type, this central sequence beingflanked in the 5' and 3' positions by α-anomeric DNA or RNA.
 13. Anoligonucleotide according to claim 11, characterized in that it isformed from a chimeric oligomer containing a surrounding centralsequence of phosphorothioate triester β-nucleoside with internucleotidelinkage of the ##STR23## type, surrounded by flanks formed fromphosphate diester alpha-nucleoside linkages.
 14. An oligonucleotideaccording to claim 12, characterized in that it is formed from achimeric oligomer containing a surrounding central sequence ofphosphorothioate triester β-nucleosides with internucleotide linkage ofthe ##STR24## type, surrounded by flanks formed from phosphate diesteralpha-nucleoside linkages.
 15. A method of preparing an oligonucleotidecomprising the general formula ##STR25## in which: Y is O or S;R₁ and R₂are respectively a residue in the 3' -O and 5' -O positions of anucleoside or of an oligonucleotide, the internucleotide linkage ofwhich is natural or modified; X is a --(CH₂)_(n) --Y¹ --W radicalwhereY¹ is S or O; each n is independently an integer from 1 to 6; and Wis either SU or --C(y²)--Z, whereU is an optionally substituted alkyl,aryl or sugar residue; Y² is O or S; and Z is an optionally substitutedalkyl, aryl or sugar residue; wherein a nucleophilic substitutionreaction by the sulfur atom of the --P--S bond of a phosphorothioatediester oligonucleotide is carried out on an alkylating agent XL, thusleading to the formation of ##STR26## phosphorothioate triesterlinkages, L being a leaving group such as halogen, ester or tosyl, and Xbeing a bioreversible group such as defined in claims 1 to
 13. 16. Amethod of preparing an oligonucleotide according to claim 1,characterized in that a nucleophilic substitution reaction by the sulfuratom of the --P--S bond of a phosphorothioate diester oligonucleotide iscarried out on an alkylating agent XL, thus leading to the formation of##STR27## phosphorothioate triester linkages, L being a leaving groupsuch as halogen, ester or tosyl, and X being a bioreversible group suchas defined in claims 1 to
 14. 17. An oligonucleotide according to claim1, characterized in that U and Z are a lower alkyl, optionallysubstituted by one or more groups chosen from amongst OH, SH and NH₂.18. An oligonucleotide according to claim 2, characterized in that U andZ are a lower alkyl, optionally substituted by one or more groups chosenfrom amongst OH, SH and NH₂.
 19. An oligonucleotide according to claim1, characterized in that R₁ and R₂ have electrically neutral sequencesat the 5' and 3' ends respectively, which are resistant to degradationby exonucleases.
 20. An oligonucleotide according to claim 2,characterized in that R₁ and R₂ have electrically neutral sequences atthe 5' and 3' ends respectively, which are resistant to degradation byexonucleases.
 21. An oligonucleotide according to claim 3, characterizedin that R₁ and R₂ have electrically neutral sequences at the 5' and 3'ends respectively, which are resistant to degradation by exonucleases.22. An oligonucleotide according to claim 5, characterized in that R₁and R₂ have electrically neutral sequences at the 5' and 3' endsrespectively, which are resistant to degradation by exonucleases.
 23. Anoligonucleotide comprising the general formula ##STR28## in which: Y isO or S;R₁ and R₂ are respectively a residue in the 3' -O and 5' -Opositions of a nucleoside or of an oligonucleotide, the internucleotidelinkage of which is natural or modified; X is a --(CH₂)_(n) --Y¹ --Wradical whereY¹ is S or O; each n is independently an integer from 1 to6; and W is either SU or --C(Y²)--Z, whereU is an optionally substitutedalkyl, aryl or sugar residue; Y² O or S; and Z is an optionallysubstituted alkyl, aryl or sugar residue; characterized in that saidoligonucleotide is formed from a chimeric oligomer comprising abeta-anomeric central DNA or RNA sequence, the internucleotide linkagesof which have the formula ##STR29## this central sequence being flankedin the 5' and 3' positions by alpha-anomeric DNA or RNA.
 24. Anoligonucleotide according to claim 1, characterized in that it is formedfrom a chimeric oligomer comprising a beta-anomeric central DNA or RNAsequence, the internucleotide linkages of which have the formula##STR30## this central sequence being flanked in the 5' and 3' positionsby alpha-anomeric DNA or RNA.
 25. An oligonucleotide according to claim2, characterized in that it is formed from a chimeric oligomercomprising a beta-anomeric central DNA or RNA sequence, theinternucleotide linkages of which have the formula ##STR31## thiscentral sequence being flanked in the 5' and 3' positions byalpha-anomeric DNA or RNA.
 26. An oligonucleotide according to claim 3,characterized in that it is formed from a chimeric oligomer comprising abeta-anomeric central DNA or RNA sequence, the internucleotide linkagesof which have the formula ##STR32## this central sequence being flankedin the 5' and 3' positions by alpha-anomeric DNA or RNA.
 27. Anoligonucleotide according to claim 5, characterized in that it is formedfrom a chimeric oligomer comprising a beta-anomeric central DNA or RNAsequence, the internucleotide linkages of which have the formula##STR33## this central sequence being flanked in the 5' and 3' positionsby alpha-anomeric DNA or RNA.
 28. An oligonucleotide according to claim1, characterized in that it is formed from a chimeric oligomercomprising a beta-anomeric central DNA or RNA sequence, theinternucleotide linkages of which have the formula ##STR34## thiscentral sequence being flanked in the 5' and 3' positions byalpha-anomeric DNA or RNA.
 29. A phosphorothioate triesteroligonucleotide comprising internucleotide linkages which have a P--Sbond wherein the S moiety thereof is protected by a group comprising acarbonyl-containing moiety; andwherein said carbonyl-containing moietyis of the formula --(CH₂)_(n) --Y¹ --W wherein:Y¹ is S or O; each n isindependently an integer from 1 to 6; and W is -C(Y²)Z where Y² is O orS; and Z is optionally substituted alkyl or a sugar residue.
 30. Anoligonucleotide according to claim 29 wherein Y¹ is O.
 31. Anoligonucleotide according to claim 29 wherein Y² is O.
 32. Anoligonucleotide according to claim 29 wherein Z is optionallysubstituted alkyl.
 33. An oligonucleotide according to claim 29 having 5to 50 nucleotides.
 34. An oligonucleotide comprising the general formula##STR35## in which: Y is O;R₁ and R₂ are, respectively, a residue in the3' -O and 5' -O positions of a nucleoside or of an oligonucleotide, theinternucleotide linkage of which is natural or modified; X is--(CH₂)_(n) --S--S--U, --(CH₂)_(n) --O--C(═Y²)--Z or --(CH₂)_(n)--S--C(═O)--Z; each n is independently an integer from 1 to 6; U is anoptionally substituted alkyl, aryl or sugar residue; Y² is O or S; and Zis optionally substituted alkyl, aryl or sugar residue.
 35. Anoligonucleotide comprising the general formula ##STR36## in which: Y isO or S;R₁ and R₂ are, respectively, a residue in the 3' -O and 5' -Opositions of a nucleoside or of an oligonucleotide, the internucleotidelinkage of which is natural or modified; X is --(CH₂)_(n) --S--S--U or--(CH₂)_(n) --O--C(═Y²)--Z; each n is independently an integer from 1 to6; U is an optionally substituted alkyl, aryl or sugar residue; Y² is Oor S; and Z is optionally substituted alkyl, aryl or sugar residue.